實(shí)驗(yàn)性肝硬化大鼠細(xì)胞因子IL18、TNFα、IFNγ的變化及意義

1、實(shí)驗(yàn)性肝硬化大鼠細(xì)胞因子IL18、TNF、IFN的變化及意義 11-01-06 11:26:00 編輯：studa20 作者：黨雙鎖，高寧，程延安，邊靜，王順達(dá)，孫明珠【摘要】 目的 探討在四氯化碳(CCl4) 復(fù)合因素誘導(dǎo)實(shí)驗(yàn)性肝硬化大鼠的形成過(guò)程中，白介素18(IL18)、腫瘤壞死因子(TNF)、干擾素(IFN)的變化及意義。方法 將80只清潔級(jí)雄性SD大鼠隨機(jī)分為正常對(duì)照組和造模2周組、4周組、6周組，每組20只。予以CCl4復(fù)合因素誘導(dǎo)大鼠肝硬化，并在2、4、6周3個(gè)時(shí)間點(diǎn)分別處死6只大鼠。采用ELISA法檢測(cè)大鼠血清IL18、TNF、IFN和肝臟組織勻漿上清液中的IL18含量；HE染

2、色觀察肝臟的組織病理學(xué)變化。結(jié)果 CCl4復(fù)合因素誘導(dǎo)的實(shí)驗(yàn)性大鼠，組織病理學(xué)觀察發(fā)現(xiàn)，2周時(shí)大鼠肝臟組織細(xì)胞腫脹變性，6周時(shí)有大量纖維增生，部分肝組織有假小葉的形成；隨著造模時(shí)間的延長(zhǎng)，實(shí)驗(yàn)大鼠血清IL18、TNF、IFN水平逐漸升高，造模6周組與正常對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)；造模組肝臟組織勻漿中IL18隨肝損害程度的加重而升高，造模6周組與正常對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義(P0.01)。結(jié)論 在CCl4復(fù)合因素誘導(dǎo)大鼠肝硬化的形成過(guò)程中，IL18、TNF、IFN水平逐漸升高，提示該3種細(xì)胞因子與肝硬化的形成及發(fā)展有關(guān)。 【關(guān)鍵詞】 大鼠；肝硬化；白介素18；腫瘤壞死因子；干擾

3、素ABSTRACT: Objective To investigate the changes of interleukin18 (IL18), tumor necrosis factor(TNF) and interferon (IFN) levels of liver cirrhosis induced by the composite factors of carbon tetrachloride (CCl4) in SD rats and their significance. Methods Totally 80 male SD rats of clean class were ra

4、ndomly divided into normal control group (20 rats) and model groups, the latter of which were further divided into three groups according to the length of administration time, namely, 2week group (2 wk group), 4week group (4 wk group) and 6week group (6 wk group), with 20 rats in each. Six rats were

5、 killed after 2 wk, 4 wk and 6 wk administration time, respectively. The rat serum levels of IL18, TNF and IFN and the hepatic homogenate supernatant of IL18 were detected by ELISA; pathological changes of liver tissues were observed by HE staining. Results Pathological observation revealed that in

6、the model groups hepatic cells degenerated and swelled at week 2 while large amounts of fibrosis and pseudolobules of some liver tissues occurred at week 6. The serum levels of IL18, TNF and IFN were gradually increased with the modeling time, and they were significantly higher in 6week group than i

7、n normal control group (P0.01). The levels of hepatic homogenate supernatant of IL18 in the model groups were elevated with liver damage, and they were significantly higher in 6week group than in normal control group (P0.01). Conclusion During the formation of liver cirrhosis induced by composite fa

8、ctors of CCl4 in rats, IL18, TNF and IFN levels gradually increase, suggesting that the three cytokines play a certain role during the occurrence of liver cirrhosis in rats.KEY WORDS: rat; liver cirrhosis; interleukin18; tumor necrosis factor (TNF); interferon (IFN)肝硬化是慢性肝損傷所共有的終末病理改變。在肝硬化的形成中，有大量細(xì)胞

9、因子通過(guò)參與調(diào)節(jié)組織細(xì)胞的損傷修復(fù)過(guò)程而發(fā)揮作用1。新近發(fā)現(xiàn)，白介素18(interleukin18, IL18)是新的致炎因子。研究發(fā)現(xiàn)，IL18參與了免疫調(diào)控，其過(guò)度表達(dá)可能引起免疫系統(tǒng)紊亂和組織器官的免疫炎性損害。本研究通過(guò)建立四氯化碳(CCl4)復(fù)合因素誘導(dǎo)的大鼠肝硬化模型，動(dòng)態(tài)檢測(cè)造模組大鼠IL18及相關(guān)細(xì)胞因子腫瘤壞死因子(TNF)、干擾素(IFN)水平的變化，初步探討細(xì)胞因子在肝硬化形成中的作用及意義。1 材料與方法1.1 主要試劑及儀器CCl4(分析純)購(gòu)自西安化學(xué)試劑廠；橄欖油(化學(xué)純)、膽固醇(分析純)購(gòu)自上?；瘜W(xué)試劑公司；大鼠IL18定量ELISA試劑盒為美國(guó)Biosou

10、rce公司生產(chǎn),上海森雄科技有限公司進(jìn)口分裝；大鼠TNF、IFN定量ELISA試劑盒購(gòu)自上海西唐生物有限公司；RIPA裂解液購(gòu)自北京索萊寶科技有限公司；DG3021型酶聯(lián)免疫檢測(cè)儀為華東電子管廠產(chǎn)品。1.2 實(shí)驗(yàn)動(dòng)物與分組清潔級(jí)雄性SD大鼠80只，34個(gè)月齡，體重360380g，平均(36714.12)g，購(gòu)于西安交通大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物中心。據(jù)肝硬化造模時(shí)間長(zhǎng)短將SD大鼠隨機(jī)分成4組：正常對(duì)照組、造模2周組、造模4周組、造模6周組，每組20只。所有大鼠分籠飼養(yǎng)于西安交通大學(xué)醫(yī)學(xué)院實(shí)驗(yàn)動(dòng)物房。正常對(duì)照組，自由取食且不做任何處理；造模2、4、6周組，每周2次皮下注射400mL/L CCl4橄欖油溶

11、液3mL/kg，首次劑量為5mL/kg；同時(shí)給予高脂飼料(80%單純玉米粉+20%豬油+0.5%膽固醇)作為唯一飲食，并以300mL/L乙醇作為飲水飼養(yǎng)動(dòng)物。每周一、四注射，且注射前稱體重。于造摸2、4、6周時(shí)各組分別隨機(jī)選取動(dòng)物6只，水合氯醛腹腔內(nèi)麻醉，腹主動(dòng)脈插管取血，并留取肝臟做組織勻漿及40g/L甲醛溶液固定，常規(guī)石蠟切片，HE染色。1.3 方法1.3.1 ELISA法測(cè)定血清IL18、TNF、IFN水平血液3000r/min離心20min，吸取血清，采用雙抗體夾心ELISA法測(cè)定，嚴(yán)格按照說(shuō)明書操作。1.3.2 ELISA法測(cè)定肝組織勻漿中IL18水平取大鼠部分肝臟組織，冰冷等滲鹽水漂洗數(shù)次后，除去血液，濾紙吸干，稱取1g肝組織，用手動(dòng)勻漿器將肝組